We will develop a suite of analytical methods to determine the activity of ectopeptidases in brain tissue. Ectopeptidases are membrane-bound peptidases facing the extracellular space. They are widely understood to act as a clearance mechanism for peptides. However, recent research points to other, more subtle and important roles for these enzymes. For example, some peptides are activated, and others have their activity altered by ectopeptidases. In stroke, neurons may be damaged. Certain peptides can protect neurons in this and other neurodegenerative conditions. We hypothesize that when neurons are damaged, ectopeptidase activity is changed in order to support the neuroprotective functions of particular neuropeptides (galanin, dynorphins and enkephalins). However, the tools that can test this idea do not exist. The methods that we will create largely focus on obtaining samples. Our experimental model is the organotypic culture of the hippocampal formation. We have learned how to withdraw fluid from the extracellular space in these preparations using electroosmotic flow. Electroosmotic flow results from the interaction of an externally applied electric field and the natural charges on the surfaces of cells. By using this approach, we can pull peptide solutions through small, few hundred <m regions of the hippocampal formation. Therefore, we can create 'maps' or 'images' of ectopeptidases activity in the hippocampal formation. We propose methods to do this efficiently and reproducibly. This technique can then be used to track ectopeptidase activity with good spatial resolution in the tissue culture over time following simulated stroke-like conditions. Another new method is specific to thiol-containing compounds, with reduced glutathione (GSH) as a target. GSH is part of an important biochemical system that protects neurons from oxidative damage. In the brain, astrocytes, a form of glial cell, provide this substance to neurons, but doing so requires an ectopeptidase called 3- glutamyltranspeptidase. We will use our new method to determine changes in the activity of that ectopeptidase in stroke-like conditions. Finally, we will test the hypothesis that the neuroprotective peptide galanin, or galanin fragments resulting from ectopeptidase activity, are neuroprotective because they discourage the inflammatory response of microglia.